Title

Author

Date of Award

Level of Access

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Benildo G. de los Reyes

Second Committee Member

Mary E. Rumpho-Kennedy

Third Committee Member

Raymond C. Fort

Abstract

Plants are either sensitive or insensitive to low temperatures. Cool-season species acclimate to chilling and develop tolerance to freezing. Warm-season species vary in the degree of sensitivity to chilling and are not capable of acclimation. Both freezing and chilling tolerance involve the activity of an intricately coordinated network of genes. The regulatory network that defines cold acclimation and freezing tolerance is well understood in Arabidopsis. The centerpiece of this network is a group of transcriptional activators that coordinate a battery of downstream defense-related genes. In contrast, little is known about the corresponding regulon in non-acclimating species. This study utilized the available tools of functional genomics and genome sequence resources of rice (Oryza sativa L.), a chilling-sensitive species, to examine the cold stress transcriptome of young seedlings of a relatively tolerant japonica cultivar (CT6748-8-CA-17). The goal was to use rice as a model towards understanding the dynamics of the early response regulatory network through a survey of gene expression changes during the critical first 24 hours of stress. The expression of 1,550 unique genes represented as spotted cDNA probes on a microarray was profiled by interrogation with a pair of control (28°C) and stressed (10°C) RNA isolated after 0.5, 2, 6, 12 and 24 hours. The expression data showed that early response involves two waves of induction and repression. The first wave started within the first 2 hours of stress, hence the genes were collectively designated as 'rapidly induced/repressed early response genes' (Group-I). The second wave did not start until after 2 hours of stress, hence the genes were collectively designated as 'delayed induced/repressed early response genes' (Group-II). The functional categories of genes overlap between Groups-I and II. In general, balanced upregulation and downregulation of genes involved in signal transduction, growth and development, metabolism, transport, protein synthesis, modification and degradation reflects the 'physiologically stressed' status of the plant, consistent with the initial reduction in growth rate. Early gene expression responses also suggest an active mechanism by which cellular resources are temporarily being redirected from growth related processes to overall physiological adjustments and early defenses. Potential regulators of early responses were also identified in the survey. The first is a novel cold stress-related bZIP transcription factor (OsbZIP) induced rapidly but transiently within the first 2 to 6 hours. Other potential early regulators are transiently expressed after 6 hours. These include a bHLH transcription (OslCE)factor similar to the ICE1 of Arabidopsis, a Myb protein similar to the previously identified cold stress response regulator Osmyb4, and a C3HC4 zinc-finger protein related to HOSl of Arabidopsis. The layered fashions by which these transcription factors are expressed suggest a mechanism for fine-tuned regulation of the early response genetic network. The transcriptome survey also suggests that ABA is not essential in the early responses and other molecules such as H2O2 are probably involved. The results of this study set the stage for future investigation of the entire pathway by a combinatorial approach that includes genome-wide transcript profiling, promoter-reporter assay, protein-DNA interaction analysis and reverse genetics.